eBook: Optimizing Gene-Editing Workflows - 13

Figure 4. Target-specific lentivirus vector.
Figure 3. TCR expression following knock-out in Cas9-expressing
Jurkat cells. Cells were electroporated with 0.1
nmol TCR sgRNA, and TCR expression was analyzed by
flow cytometry 72 hours later. Parental cells: blue; TCR
knock-out cell pool: green.
ed cells, therefore these lentiviruses require only
a Biosafety Level 2 facility. The particles contain a
Cas9-NSL-Flag gene driven by an EF1a promoter
along with an antibiotic selection marker, typically
hygromycin or puromycin.
The integrating Cas9 lentivirus can be used to generate
Cas9 expressing cells, which can then be transduced
or electroporated with sgRNAs targeting a
gene of interest to quickly achieve the desired genetic
modification.
Pre-validated lentiviruses allow for dual expression
of Cas9 and sgRNAs. These integrating lentiviruses
contain a Cas9-NLS-Flag gene driven by an EF1a promoter,
along with 4 sgRNAs driven by a U6 promoter
and a puromycin selection marker (Figure 4). Puromycin
selection forces integration of the construct
into the genome and results in sustained expression
of both Cas9 and sgRNA, which increases knockdown
efficiency.
Figure 5. Knock-down of LAG3. LAG3 overexpressing Jurkat
cells (BPS Bioscience, #79813) were transduced via
spinoculation with LAG3 CRISPR/Cas9 lentivirus (BPS Bioscience,
#78053). 72 hours after transduction, cells were
stained with PE anti-human LAG3 antibody and analyzed
by flow cytometry. M3: population of cells in which
LAG3 is knocked-down.
Figure 5 illustrates the transient knock-down efficiency
in LAG3-overexpressing Jurkat cells transduced
with Cas9 and LAG3-targeting sgRNAs.
However, integration into the genome occurs randomly
and may occasionally disrupt a biologically
13
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